Pepper Hybrid &#39;E 499526&#39;

ABSTRACT

Hybrid pepper cultivar designated ‘E 499526’ which is a Sweetbite type and suitable for covered cultivation and open field, is disclosed. The invention relates to the seeds of hybrid pepper cultivar ‘E 499526’ and to the plants of hybrid pepper cultivar ‘E 499526’. The invention also relates to methods for producing a pepper plant, either inbred or hybrid, by crossing the hybrid cultivar ‘E 499526’ with itself or another pepper cultivar. The invention further relates to methods for producing other pepper cultivars derived from the hybrid ‘E 499526’.

BACKGROUND OF THE INVENTION

The present invention relates to a new and distinctive pepper (Capsicumannuum) hybrid designated ‘E 499526’. Pepper hybrid ‘E 499526’ producesfruit which is very small jalapeno pepper size) but mild. In addition,the plants are suitable for covered cultivation and open field growing.All publications cited in this application are herein incorporated byreference.

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possess the traits to meetthe program goals. The selected germplasm is crossed in order torecombine the desired traits and through selection varieties or parentlines are developed. The goal is to combine in a single variety orhybrid an improved combination of desirable traits from the parentalgermplasm. These important traits may include higher yield, fieldperformance, fruit and agronomic quality such as fruit shape and length,resistance to diseases and insects, and tolerance to drought and heat.

The bell pepper (Capsicum annuum) originated in Mexico and theneighboring areas of Central America. Soon after Columbus’ discovery ofthis plant, it was grown worldwide and used as a spice and a medicine.Today, pepper plants can be found growing wild in tropical areas aroundthe world. Many countries grow it as a crop. Many of the hot peppers canbe found in Latin America and China, but the US prefers sweet bellpeppers. Peppers are used for fresh consumption, and they are processedinto powders, sauces, and salsas. Many of the new cultivars grown todaycan be traced back to the early plants.

The genus Capsicum and species annuum includes most of the peppers grownin the U.S. These can be further grouped into two broad categories:chile peppers which are pungent (hot) and sweet peppers which arenon-pungent (mild). The U.S. produces 4 percent of the world's capsicumpeppers (sweet and hot), ranking sixth behind China, Mexico, Turkey,Spain and Nigeria. Bell peppers are the most common sweet pepper and arefound in virtually every retail produce department. Grown commerciallyin most states, the U.S. industry in largely concentrated in Californiaand Florida, which together accounted for 78% of output in 2000. NewJersey, Georgia and North Carolina round out the top five producingstates. (Economic Research Service, USDA. Vegetables and MelonsOutlook/VGS-288/Dec. 14, 2001.)

Bell peppers are eaten raw, cooked, immature and mature. Oftennutritional content is altered by the changes in the way they areconsumed. Per capita consumption of bell peppers in 1995 was 6.2 pounds.They are an excellent source of Vitamin C, Vitamin A, and Calcium. Redpeppers have more of these qualities than the immature green peppers.

Peppers grown in temperate regions are herbaceous annuals, but areherbaceous perennials where temperatures do not drop below freezing.Pepper plants' growth habit may be prostrate, compact, or erect, but itis determinate in that after it produces nine to eleven leaves a singlestem terminates in flowers. These plants are grown for the edible fleshyfruit produced by this dichotomous growth. Peppers are non-climactericwhich means they do not produce ethylene. They need to stay on the vineto continue the ripening process. A deep taproot will form if the plantroot system is uninjured during transplanting. The spindle root willdevelop fibrous secondary root systems spreading laterally and downward.On the soil surface the stem will produce adventitious roots, but not aseasily as tomatoes. The leaves of the pepper plant arise singly and aresimple, entire, and asymmetrical. Typical of all Solanaceous plants, theleaves are arranged alternately on the stem. They are shiny and glabrousand vary in shape from broadly ovate to ovate lanceolate. The flowersdevelop singly or in twos or threes continuously as the upper structureof the plant proliferates. The corolla is white and five lobed while theanthers are bluish or yellowish in color. The flowers have an openanther formation and will indefinitely self-pollinate. They are alsopollinated by insects, which increases the chances of cross-pollination.Unlike tomatoes, whose pollen becomes nonviable in high temperatures,the pepper flowers’ pollen is not extremely heat sensitive and itremains viable up to 1000 Fahrenheit producing fruit throughout theseason.

The fruit of a pepper plant is classified as a berry with colors fromgreen, yellow, red, purple, black, brown, white and orange. Green is animmature fruit, yet commonly eaten this way, and as the fruit matures itchanges color. In most commercial cultivars color changes are from greento red, green to yellow or green to orange. Usually, fruits of thepurple and white varieties have these colors as they develop, andtherefore do not have a green stage. For fruit to set, the ovaries needto be fertilized. Auxin is then produced by the seeds, which determinefruit cell elongation. The number of seeds fertilized will determine thesize and shape of the fruit. The seed develop on the interior and attachto the veins. Fully developed seed is kidney shaped. There are about4,500 seeds per ounce.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, purelinecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, and recurrent selection.

The complexity of inheritance influences choice of the breeding method.Backcross breeding is used to transfer one or a few favorable genes fora highly heritable trait into a desirable cultivar. This approach hasbeen used extensively for breeding disease-resistant cultivars. Variousrecurrent selection techniques are used to improve quantitativelyinherited traits controlled by numerous genes. The use of recurrentselection in self-pollinating crops depends on the ease of pollination,the frequency of successful hybrids from each pollination, and thenumber of hybrid offspring from each successful cross.

Each breeding program should include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard,overall value of the advanced breeding lines, and number of successfulcultivars produced per unit of input (e.g., per year, per dollarexpended, etc.).

Promising advanced breeding lines are thoroughly tested and compared toappropriate standards in environments representative of the commercialtarget area(s) for three years at least. The best lines are candidatesfor new commercial cultivars; those still deficient in a few traits areused as parents to produce new populations for further selection.

These processes, which lead to the final step of marketing anddistribution, usually take from five to ten years from the time thefirst cross or selection is made. Therefore, development of newcultivars is a time-consuming process that requires precise forwardplanning, efficient use of resources, and a minimum of changes indirection.

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

The goal of pepper plant breeding is to develop new, unique and superiorpepper cultivars. The breeder initially selects and crosses two or moreparental lines, followed by repeated selfing and selection, producingmany new genetic combinations. The breeder can theoretically generatebillions of different genetic combinations via crossing, selfing andmutations. The breeder has no direct control at the cellular level.Therefore, two breeders will never develop the same line having the samepepper traits.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under different geographical,climatic and soil conditions, and further selections are then made,during and at the end of the growing season. The cultivars that aredeveloped are unpredictable. This unpredictability is because thebreeder's selection occurs in unique environments, with no control atthe DNA level (using conventional breeding procedures), and withmillions of different possible genetic combinations being generated. Abreeder of ordinary skill in the art cannot predict the final resultinglines he develops, except possibly in a very gross and general fashion.The same breeder cannot produce the same line twice by using the exactsame original parents and the same selection techniques. Thisunpredictability results in the expenditure of large research monies todevelop superior pepper cultivars.

The development of commercial pepper cultivars requires the developmentof pepper parental lines, the crossing of these lines, and theevaluation of the crosses. Pedigree breeding and recurrent selectionbreeding methods are used to develop cultivars from breedingpopulations. Breeding programs combine desirable traits from two or morevarieties or various broad-based sources into breeding pools from whichlines are developed by selfing and selection of desired phenotypes. Thenew lines are crossed with other lines and the hybrids from thesecrosses are evaluated to determine which have commercial potential.

Pedigree breeding is used commonly for the improvement ofself-pollinating crops or inbred lines of cross-pollinating crops. Twoparents which possess favorable, complementary traits are crossed toproduce an F₁. An F₂ population is produced by selfing one or severalF₁s or by intercrossing two F₁s (sib mating). Selection of the bestindividuals is usually begun in the F₂ population; then, beginning inthe F₃, the best individuals in the best families are selected.Replicated testing of families, or hybrid combinations involvingindividuals of these families, often follows in the F₄ generation toimprove the effectiveness of selection for traits with low heritability.At an advanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease as new cultivars.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror line that is the recurrent parent. The source of the trait to betransferred is called the donor parent. The resulting plant is expectedto have the attributes of the recurrent parent (e.g., cultivar) and thedesirable trait transferred from the donor parent. After the initialcross, individuals possessing the phenotype of the donor parent areselected and repeatedly crossed (backcrossed) to the recurrent parent.The resulting plant is expected to have the attributes of the recurrentparent (e.g., cultivar) and the desirable trait transferred from thedonor parent.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F₂ plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

In addition to phenotypic observations, the genotype of a plant can alsobe examined. There are many laboratory-based techniques available forthe analysis, comparison and characterization of plant genotype; amongthese are Isozyme Electrophoresis, Restriction Fragment LengthPolymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs),Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA AmplificationFingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs),Amplified Fragment Length polymorphisms (AFLPs), Simple Sequence Repeats(SSRs—which are also referred to as Microsatellites), and SingleNucleotide Polymorphisms (SNPs).

Isozyme Electrophoresis and RFLPs have been widely used to determinegenetic composition. Shoemaker and Olsen, (Molecular Linkage Map ofSoybean (Glycine max) p 6.131-6.138 in S. J. O'Brien (ed) Genetic Maps:Locus Maps of Complex Genomes, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., (1993)) developed a molecular genetic linkage mapthat consisted of 25 linkage groups with about 365 RFLP, 11 RAPD, threeclassical markers and four isozyme loci. See also, Shoemaker, R. C.,RFLP Map of Soybean, p 299-309, in Phillips, R. L. and Vasil, I. K.,eds. DNA-Based Markers in Plants, Kluwer Academic Press, Dordrecht, theNetherlands (1994).

SSR technology is currently the most efficient and practical markertechnology; more marker loci can be routinely used and more alleles permarker locus can be found using SSRs in comparison to RFLPs. Forexample, Diwan and Cregan described a highly polymorphic microsatellitelocus in soybean with as many as 26 alleles. (Diwan, N. and Cregan, P.B., Theor. Appl. Genet 95:22-225,1997.) SNPs may also be used toidentify the unique genetic composition of the invention and progenyvarieties retaining that unique genetic composition. Various molecularmarker techniques may be used in combination to enhance overallresolution.

Molecular markers, which include markers identified through the use oftechniques such as Isozyme Electrophoresis, RFLPs, RAPDs, AP-PCR, DAF,SCARs, AFLPs, SSRs, and SNPs, may be used in plant breeding. One use ofmolecular markers is Quantitative Trait Loci (QTL) mapping. QTL mappingis the use of markers which are known to be closely linked to allelesthat have measurable effects on a quantitative trait. Selection in thebreeding process is based upon the accumulation of markers linked to thepositive effecting alleles and/or the elimination of the markers linkedto the negative effecting alleles from the plant's genome.

Molecular markers can also be used during the breeding process for theselection of qualitative traits. For example, markers closely linked toalleles or markers containing sequences within the actual alleles ofinterest can be used to select plants that contain the alleles ofinterest during a backcrossing breeding program. The markers can also beused to select toward the genome of the recurrent parent and against themarkers of the donor parent. This procedure attempts to minimize theamount of genome from the donor parent that remains in the selectedplants. It can also be used to reduce the number of crosses back to therecurrent parent needed in a backcrossing program. The use of molecularmarkers in the selection process is often called genetic marker enhancedselection or marker-assisted selection. Molecular markers may also beused to identify and exclude certain sources of germplasm as parentalvarieties or ancestors of a plant by providing a means of trackinggenetic profiles through crosses.

Mutation breeding is another method of introducing new traits intopepper varieties. Mutations that occur spontaneously or are artificiallyinduced can be useful sources of variability for a plant breeder. Thegoal of artificial mutagenesis is to increase the rate of mutation for adesired characteristic. Mutation rates can be increased by manydifferent means including temperature, long-term seed storage, tissueculture conditions, radiation (such as X-rays, Gamma rays, neutrons,Beta radiation, or ultraviolet radiation), chemical mutagens (such asbase analogs like 5-bromo-uracil), antibiotics, alkylating agents (suchas sulfur mustards, nitrogen mustards, epoxides, ethyleneamines,sulfates, sulfonates, sulfones, or lactones), azide, hydroxylamine,nitrous acid or acridines. Once a desired trait is observed throughmutagenesis the trait may then be incorporated into existing germplasmby traditional breeding techniques. Details of mutation breeding can befound in Principles of Cultivar Development by Fehr, MacmillanPublishing Company, 1993.

The production of double haploids can also be used for the developmentof homozygous lines in a breeding program. Double haploids are producedby the doubling of a set of chromosomes from a heterozygous plant toproduce a completely homozygous individual. For example, see Wan et al.,Theor. Appl. Genet., 77:889-892, 1989.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Principles of Plant Breeding John Wiley and Son, pp.115-161, 1960; Allard, 1960; Simmonds, 1979; Sneep et al., 1979; Fehr,1987; “Carrots and Related Vegetable Umbelliferae”, Rubatzky, V. E., etal., 1999).

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current cultivars. In addition toshowing superior performance, there must be a demand for a new cultivarthat is compatible with industry standards or which creates a newmarket. The introduction of a new cultivar will incur additional coststo the seed producer, the grower, processor and consumer for specialadvertising and marketing, altered seed and commercial productionpractices, and new product utilization. The testing preceding release ofa new cultivar should take into consideration research and developmentcosts as well as technical superiority of the final cultivar. Forseed-propagated cultivars, it must be feasible to produce seed easilyand economically.

Pepper is an important and valuable field crop. Thus, a continuing goalof plant breeders is to develop stable, high yielding pepper hybridsthat are agronomically sound. The reasons for this goal are obviously tomaximize the amount of fruit produced on the land used as well as toimprove the fruit agronomic qualities. To accomplish this goal, thepepper breeder must select and develop pepper plants that have thetraits that result in superior parental lines for producing hybrids.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described inconjunction with systems, tools, and methods which are meant to beexemplary and illustrative, not limiting in scope. In variousembodiments, one or more of the above-described problems have beenreduced or eliminated, while other embodiments are directed to otherimprovements.

According to the invention, there is provided a hybrid pepper designated‘E 499526’. This invention thus relates to the seeds of hybrid pepper ‘E499526’, to the plants of pepper ‘E 499526’ and to methods for producinga pepper plant produced by crossing the hybrid cultivar ‘E 499526’ withitself or another pepper cultivar, and to methods for producing a pepperplant containing in its genetic material one or more transgenes and tothe transgenic pepper plants produced by that method. This inventionalso relates to methods for producing other pepper cultivars derivedfrom hybrid pepper cultivar ‘E 499526’ and to the pepper cultivarsderived by the use of those methods. This invention further relates topepper seeds and plants produced by crossing the hybrid cultivar ‘E499526’ with another pepper cultivar.

Parts of the pepper plant ‘E 499526’ are also provided, such as e.g.,fruits, leaves, stems, flowers, pollen and ovules.

In another aspect, the present invention provides regenerable cells foruse in tissue culture of pepper plant ‘E 499526’. The tissue culturewill preferably be capable of regenerating plants having thephysiological and morphological characteristics of the foregoing pepperplant, and of regenerating plants having substantially the same genotypeas the foregoing inbred pepper plant. Preferably, the regenerable cellsin such tissue cultures will be produced from embryo, protoplast,meristematic cell, callus, pollen, leaf, stem, petiole, root, root tip,fruit, seed, flower, anther, pistil or the like. Still further, thepresent invention provides pepper plants regenerated from tissuecultures of the invention.

Another aspect of the invention is to provide methods for producingother pepper plants derived from pepper cultivar ‘E 499526’. Peppercultivars derived by the use of those methods are also part of theinvention.

In another aspect, the present invention provides for single geneconverted plants of ‘E 499526’. The single transferred gene maypreferably be a dominant or recessive allele. Preferably, the singletransferred gene will confer such trait as sex determination, herbicideresistance, insect resistance, resistance for bacterial, fungal, orviral disease, improved harvest characteristics, enhanced nutritionalquality, or improved agronomic quality. The single gene may be anaturally occurring pepper gene or a transgene introduced throughgenetic engineering techniques.

The invention further provides methods for developing pepper plants in apepper plant breeding program using plant breeding techniques includingrecurrent selection, backcrossing, pedigree breeding, restrictionfragment length polymorphism enhanced selection, genetic marker enhancedselection and transformation. Marker loci such as restriction fragmentpolymorphisms or random amplified DNA have been published for many yearsand may be used for selection (See Pierce et al., HortScience (1990)25:605-615, Wehner, T., Cucurbit Genetics Cooperative Report, (1997) 20:66-88 and Kennard et al., Theorical Applied Genetics (1994) 89:217-224).Seeds, pepper plants, and parts thereof produced by such breedingmethods are also part of the invention.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by reference bystudy of the following descriptions.

DEFINITIONS

In the description and tables that follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Allele. “Allele” means any of one or more alternative forms of a gene,all of which relate to one trait or characteristic. In a diploid cell ororganism, the two alleles of a given gene occupy corresponding loci on apair of homologous chromosomes.

Backcrossing. “Backcrossing” means a process in which a breederrepeatedly crosses hybrid progeny back to one of the parents, forexample, a first generation hybrid F₁ with one of the parental genotypeof the F₁ hybrid.

Covered cultivation. Any type of cultivation where the plants are notexposed to direct sunlight. The covering includes but is not limited togreenhouses, glasshouses, net-houses, plastic houses and tunnels.

Essentially all the physiological and morphological characteristics. Aplant having essentially all the physiological and morphologicalcharacteristics means a plant having the physiological and morphologicalcharacteristics of the recurrent parent, except for the characteristicsderived from the converted gene.

Internode. An “internode” means the stem segment between nodes.

Propagate. To “propagate” a plant means to reproduce the plant by meansincluding, but not limited to, seeds, cuttings, divisions, tissueculture, embryo culture or other in vitro method.

Quantitative Trait Loci (QTL). “Quantitative trait loci” refer togenetic loci that control to some degree numerically representabletraits that are usually continuously distributed.

Regeneration. “Regeneration” refers to the development of a plant fromtissue culture.

Single gene converted. “Single gene converted” or conversion plantrefers to plants which are developed by a plant breeding techniquecalled backcrossing wherein essentially all of the desired morphologicaland physiological characteristics of an inbred are recovered in additionto the single gene transferred into the inbred via the backcrossingtechnique or via genetic engineering.

Sweetbite. “Sweetbite” means any sweet pepper plant having fruit with 2or 3 locules, conical shaped, with a diameter between 1.5 centimetersand 3 centimeters and a length between 3 centimeters and 7 centimeters.

Transgene. A “transgene” is a gene taken or copied from one organism andinserted into another organism. A transgene may be a gene that isforeign to the receiving organism or it may be a modified version of anative, or endogenous, gene.

DETAILED DESCRIPTION OF THE INVENTION

‘E 499526’ is a hybrid pepper cultivar with high yield potential. It isa medium vigorous plant that produces medium green immature fruit andlight yellow mature fruit of a very small size jalapeno pepper size) butmild flavor (not hot). In addition, the plants are suitable for coveredcultivation and open field growing. The hybrid cultivar has shownuniformity and stability for the traits, within the limits ofenvironmental influence for the traits. The cultivar has been increasedwith continued observation for uniformity. No variant traits have beenobserved or are expected in ‘E 499526’.

Hybrid pepper cultivar ‘E 499526’ has the following morphologic andother characteristics.

TABLE 1 VARIETY DESCRIPTION INFORMATION FOR E 499526 GENERAL: Type:Sweetbite Usage: Fresh market Type of culture: Covered cultivation andopen field PLANT: Seedling, anthocyanin coloration of hypocotyl: PresentShortened internode: Absent Vigor: Medium Flower, attitude of peduncle:Non-erect FRUIT: Color before maturity: Green Intensity of color beforematurity: Medium Length: 4 cm Diameter: 2 cm Predominant shape oflongitundinal section: Trapezoid Color at maturity: Yellow Intensity ofcolor at maturity: Light Predominant number of locules: Two and threeCapsaicin in placenta: Absent Time of ripening (color change of fruitson 50% of plants): Medium DISEASE Tobamovirus pathotype P₀: ResistantRESISTANCE: Tobamovirus pathotype P₁: Susceptible Tobamovirus pathotypeP₁₋₁: Susceptible Tobamovirus pathotype P₁₋₂₋₃: Susceptible Potato VirusY pathotype P₀: Susceptible Potato Virus Y pathotype P₁: SusceptiblePotato Virus Y pathotype P₁₋₂: Susceptible Phytophthora capsici:Susceptible Tomato Spotted Wilt Virus: Susceptible Cucumber MosaicVirus: Susceptible

FURTHER EMBODIMENTS OF THE INVENTION

This invention also is directed to methods for producing a pepper plantby crossing a first parent pepper plant with a second parent pepperplant wherein either the first or second parent pepper plant is a hybridpepper plant of the cultivar ‘E 499526’. Further, both first and secondparent pepper plants can come from the hybrid pepper cultivar ‘E499526’. All plants produced using hybrid pepper cultivar ‘E 499526’ asa parent are within the scope of this invention, including plantsderived from hybrid pepper cultivar ‘E 499526’.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which pepper plants can be regenerated,plant calli, plant clumps and plant cells that are intact in plants orparts of plants, such as embryos, pollen, ovules, flowers, leaves,stems, and the like.

As it is well known in the art, tissue culture of pepper can be used forthe in vitro regeneration of pepper plants. Tissues cultures of varioustissues of pepper and regeneration of plants therefrom are well knownand published. By way of example, tissue cultures, some comprisingorgans to be used to produce regenerated plants, have been described inBurza et al., Plant Breeding. 1995, 114: 4, 341-345, Pellinen,Angewandte Botanik. 1997, 71: 3/4,116-118, Kuijpers et al., Plant CellTissue and Organ Culture. 1996, 46: 1, 81-83, Colijn-Hooymans et al.,Plant Cell Tissue and Organ Culture. 1994, 39: 3, 211-217, Lou et al.,HortScience. 1994, 29: 8, 906-909, Tabei et al., Breeding Science. 1994,44: 1, 47-51, Sarmanto et al., Plant Cell Tissue and Organ Culture 31:3185-193 (1992), Cade et al., Journal of the American Society forHorticultural Science 115:4 691-696 (1990), Chee et al., HortScience25:7, 792-793 (1990), Kim et al., HortScience 24:4 702 (1989), Punja etal., Plant Cell Report 9:2 61-64 (1990). Pepper plants could beregenerated by somatic embryogenesis. It is clear from the literaturethat the state of the art is such that these methods of obtaining plantsare “conventional” in the sense that they are routinely used and have avery high rate of success. Thus, another aspect of this invention is toprovide cells which upon growth and differentiation produce pepperplants having the physiological and morphological characteristics ofhybrid pepper cultivar ‘E 499526’.

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreignadditional and/or modified genes are referred to herein collectively as“transgenes”. Over the last fifteen to twenty years several methods forproducing transgenic plants have been developed, and the presentinvention, in particular embodiments, also relates to transformedversions of the claimed cultivar.

Plant transformation involves the construction of an expression vectorthat will function in plant cells. Such a vector comprises DNAcomprising a gene under control of or operatively linked to a regulatoryelement (for example, a promoter). The expression vector may contain oneor more such operably linked gene/regulatory element combinations. Thevector(s) may be in the form of a plasmid, and can be used alone or incombination with other plasmids, to provide transformed pepper plants,using transformation methods as described below to incorporatetransgenes into the genetic material of the pepper plant(s).

Expression Vectors for Pepper Transformation: Marker Genes

Expression vectors include at least one genetic marker, operably linkedto a regulatory element (a promoter, for example) that allowstransformed cells containing the marker to be either recovered bynegative selection, i.e., inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or a herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene, isolated from transposonTn5, which when placed under the control of plant regulatory signalswhich confers resistance to kanamycin (Fraley et al., Proc. Natl. Acad.Sci. U.S.A., 80:4803 (1983)). Another commonly used selectable markergene is the hygromycin phosphotransferase gene which confers resistanceto the antibiotic hygromycin (Vanden Elzen et al., Plant Mol. Biol.,5:299 (1985)).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase,the bleomycin resistance determinant (Hayford et al., Plant Physiol.86:1216 (1988), Jones et al., Mol. Gen. Genet., 210:86 (1987), Svab etal., Plant Mol. Biol. 14:197 (1990), Hille et al., Plant Mol. Biol.7:171 (1986)). Other selectable marker genes confer resistance toherbicides such as glyphosate, glufosinate or bromoxynil (Comai et al.,Nature 317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618(1990) and Stalker et al., Science 242:419-423 (1988)).

Selectable marker genes for plant transformation that are not ofbacterial origin include, for example, mouse dihydrofolate reductase,plant 5-enolpyruvylshikimate-3-phosphate synthase and plant acetolactatesynthase (Eichholtz et al., Somatic Cell Mol. Genet. 13:67 (1987), Shahet al., Science 233:478 (1986), Charest et al., Plant Cell Rep. 8:643(1990)).

Another class of marker genes for plant transformation requiresscreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include α-glucuronidase (GUS),α-galactosidase, luciferase and chloramphenicol, acetyltransferase(Jefferson, R. A., Plant Mol. Biol. Rep. 5:387 (1987), Teeri et al.,EMBO J. 8:343 (1989), Koncz et al., Proc. Natl. Acad. Sci U.S.A. 84:131(1987), DeBlock et al., EMBO J. 3:1681 (1984)).

In vivo methods for visualizing GUS activity that do not requiredestruction of plant tissues are available (Molecular Probes publication2908, IMAGENE GREEN, p. 1-4 (1993) and Naleway et al., J. Cell Biol.115:151 a (1991)). However, these in vivo methods for visualizing GUSactivity have not proven useful for recovery of transformed cellsbecause of low sensitivity, high fluorescent backgrounds and limitationsassociated with the use of luciferase genes as selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has beenutilized as a marker for gene expression in prokaryotic and eukaryoticcells (Chalfie et al., Science 263:802 (1994)). GFP and mutants of GFPmay be used as screenable markers.

Expression Vectors for Pepper Transformation: Promoters

Genes included in expression vectors must be driven by a nucleotidesequence comprising a regulatory element, for example, a promoter.Several types of promoters are now well known in the transformationarts, as are other regulatory elements that can be used alone or incombination with promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred”.Promoters which initiate transcription only in certain tissue arereferred to as “tissue-specific”. A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter which is active under mostenvironmental conditions.

A. Inducible Promoters

An inducible promoter is operably linked to a gene for expression inpepper. Optionally, the inducible promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in pepper. With an inducible promoter the rateof transcription increases in response to an inducing agent.

Any inducible promoter can be used in the instant invention. See Ward etal., Plant Mol. Biol. 22:361-366 (1993). Exemplary inducible promotersinclude, but are not limited to, that from the ACEI system whichresponds to copper (Meft et al., Proc. Natl. Acad. Sci U.S.A.90:4567-4571 (1993)); In2 gene from maize which responds tobenzenesulfonamide herbicide safeners (Hershey et al., Mol. Gen Genetics227:229-237 (1991) and Gatz et al., Mol. Gen. Genetics 243:32-38 (1994))or Tet repressor from Tn10 (Gatz et al., Mol. Gen. Genetics 227:229-237(1991). A particularly preferred inducible promoter is a promoter thatresponds to an inducing agent to which plants do not normally respond.An exemplary inducible promoter is the inducible promoter from a steroidhormone gene, the transcriptional activity of which is induced by aglucocorticosteroid hormone. Schena et al., Proc. Natl. Acad. Sci.U.S.A. 88:0421 (1991).

B. Constitutive Promoters

A constitutive promoter is operably linked to a gene for expression inpepper or the constitutive promoter is operably linked to a nucleotidesequence encoding a signal sequence which is operably linked to a genefor expression in pepper.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell et al., Nature 313:810-812 (1985) and the promoters from suchgenes as rice actin (McElroy et al., Plant Cell 2:163-171 (1990));ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) andChristensen et al., Plant Mol. Biol. 18:675-689 (1992)); PEMU (Last etal., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten et al., EMBO J.3:2723-2730 (1984)) and maize H3 histone (Lepetit et al., Mol. Gen.Genetics 231:276-285 (1992) and Atanassova et al., Plant Journal 2 (3):291-300 (1992)). The ALS promoter, Xba1/NcoI fragment 5′ to the Brassicanapus ALS3 structural gene (or a nucleotide sequence similarity to saidXba1/NcoI fragment), represents a particularly useful constitutivepromoter. See PCT application WO 96/30530.

C. Tissue-specific or Tissue-preferred Promoters

A tissue-specific promoter is operably linked to a gene for expressionin pepper. Optionally, the tissue-specific promoter is operably linkedto a nucleotide sequence encoding a signal sequence which is operablylinked to a gene for expression in pepper. Plants transformed with agene of interest operably linked to a tissue-specific promoter producethe protein product of the transgene exclusively, or preferentially, ina specific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promoter,such as that from the phaseolin gene (Murai et al., Science 23:476-482(1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. U.S.A.82:3320-3324 (1985)); a leaf-specific and light-induced promoter such asthat from cab or rubisco (Simpson et al., EMBO J. 4(11):2723-2729 (1985)and Timko et al., Nature 318:579-582 (1985)); an anther-specificpromoter such as that from LAT52 (Twell et al., Mol. Gen. Genetics217:240-245 (1989)); a pollen-specific promoter such as that from Zm13(Guerrero et al., Mol. Gen. Genetics 244:161-168 (1993)) or amicrospore-preferred promoter such as that from apg (Twell et al., Sex.Plant Reprod. 6:217-224 (1993)).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall ormitochondrion or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample Becker et al., Plant Mol. Biol. 20:49 (1992), Close, P. S.,Master's Thesis, Iowa State University (1993), Knox, C., et al.,“Structure and Organization of Two Divergent Alpha-Amylase Genes fromBarley”, Plant Mol. Biol. 9:3-17 (1987), Lerner et al., Plant Physiol.91:124-129 (1989), Fontes et al., Plant Cell 3:483-496 (1991), Matsuokaet al., Proc. Nat. Acad. Sci. 88:834 (1991), Gould et al., J. Cell.Biol. 108:1657 (1989), Creissen et al., Plant J. 2:129 (1991), Kalderon,et al., A short amino acid sequence able to specify nuclear location,Cell 39:499-509 (1984), Steifel, et al., Expression of a maize cell wallhydroxyproline-rich glycoprotein gene in early leaf and root vasculardifferentiation, Plant Cell 2:785-793 (1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal Biochem. 114:92-6(1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is pepper. In another preferredembodiment, the biomass of interest is seed. For the relatively smallnumber of transgenic plants that show higher levels of expression, agenetic map can be generated, primarily via conventional RFLP, PCR andSSR analysis, which identifies the approximate chromosomal location ofthe integrated DNA molecule. For exemplary methodologies in this regard,see Glick and Thompson, Methods in Plant Molecular Biology andBiotechnology, CRC Press, Boca Raton 269:284 (1993). Map informationconcerning chromosomal location is useful for proprietary protection ofa subject transgenic plant. If unauthorized propagation is undertakenand crosses made with other germplasm, the map of the integration regioncan be compared to similar maps for suspect plants, to determine if thelatter have a common parentage with the subject plant. Map comparisonswould involve hybridizations, RFLP, PCR, SSR and sequencing, all ofwhich are conventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

1. Genes That Confer Resistance to Pests or Disease and That Encode:

A. Plant disease resistance genes. Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant line can be transformed with a clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example Jones et al., Science 266:789 (1994)(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin et al., Science 262:1432 (1993) (tomato Pto gene for resistanceto Pseudomonas syringae pv. tomato encodes a protein kinase); Mindrinoset al., Cell 78:1089 (1994) (Arabidopsis RSP2 gene for resistance toPseudomonas syringae).

B. A Bacillus thuringiensis protein, a derivative thereof or a syntheticpolypeptide modeled thereon. See, for example, Geiser et al., Gene48:109 (1986), who disclose the cloning and nucleotide sequence of a Btδ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxin genes canbe purchased from American Type Culture Collection, Manassas, Va., forexample, under ATCC Accession Nos. 40098, 67136, 31995 and 31998.

C. A lectin. See, for example, the disclosure by Van Damme et al., PlantMolec. Biol. 24:25 (1994), who disclose the nucleotide sequences ofseveral Clivia miniata mannose-binding lectin genes.

D. A vitamin-binding protein such as avidin. See PCT application US93/06487, the contents of which are hereby incorporated by reference.The application teaches the use of avidin and avidin homologues aslarvicides against insect pests.

E. An enzyme inhibitor, for example, a protease or proteinase inhibitoror an amylase inhibitor. See, for example, Abe et al., J. Biol. Chem.262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor), Huub et al., Plant Molec. Biol. 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor I), Sumitani etal., Biosci. Biotech. Biochem. 57:1243 (1993) (nucleotide sequence ofStreptomyces nitrosporeus α-amylase inhibitor).

F. An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

G. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem. 269:9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor), and Pratt etal., Biochem. Biophys. Res. Comm. 163:1243 (1989) (an allostatin isidentified in Diploptera puntata). See also U.S. Pat. No. 5,266,317 toTomalski et al., who disclose genes encoding insect-specific, paralyticneurotoxins.

H. An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see Pang et al., Gene 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

I. An enzyme responsible for a hyper-accumulation of a monoterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

J. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hornworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21:673 (1993), who provide the nucleotide sequence ofthe parsley ubi4-2 polyubiquitin gene.

K. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24:757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., Plant Physiol. 104:1467 (1994), who provide the nucleotide sequenceof a maize calmodulin cDNA clone.

L. A hydrophobic moment peptide. See PCT application WO 95/16776(disclosure of peptide derivatives of tachyplesin which inhibit fungalplant pathogens) and PCT application WO 95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance), the respectivecontents of which are hereby incorporated by reference.

M. A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure of Jaynes et al., Plant Sci 89:43 (1993), ofheterologous expression of a cecropin-β, lytic peptide analog to rendertransgenic tobacco plants resistant to Pseudomonas solanacearum.

N. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. rev. Phytopathol.28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus and tobacco mosaic virus. Id.

O. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. SeeTaylor et al., Abstract #497, Seventh Int'l Symposium on MolecularPlant-Microbe Interactions (Edinburgh, Scotland) (1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

P. A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

Q. A developmental-arrestive protein produced in nature by a pathogen ora parasite. Thus, fungal endo-α-1,4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1,4-D-galacturonase. See Lamb et al., Bio/Technology10:1436 (1992). The cloning and characterization of a gene which encodesa bean endopolygalacturonase-inhibiting protein is described by Toubartet al., Plant J. 2:367 (1992).

R. A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bio/Technology 10:305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

2. Genes That Confer Resistance to an Herbicide:

A. An herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl. Genet 80:449(1990), respectively.

B. Glyphosate (resistance conferred by mutant5-enolpyruvlshikimate-3-phosphate synthase (EPSPS) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase (PAT) and Streptomyceshygroscopicus phosphinothricin-acetyl transferase PAT bar genes), andpyridinoxy or phenoxy proprionic acids and cyclohexones (ACCaseinhibitor-encoding genes). See, for example, U.S. Pat. No. 4,940,835 toShah, et al., which discloses the nucleotide sequence of a form of EPSPSwhich can confer glyphosate resistance. A DNA molecule encoding a mutantaroA gene can be obtained under ATCC accession number 39256, and thenucleotide sequence of the mutant gene is disclosed in U.S. Pat. No.4,769,061 to Comai. See also Umaballava-Mobapathie in TransgenicResearch. 1999, 8: 1, 33-44 that discloses Lactuca sativa resistant toglufosinate. European patent application No. 0 333 033 to Kumada et al.,and U.S. Pat. No. 4,975,374 to Goodman et al., disclose nucleotidesequences of glutamine synthetase genes which confer resistance toherbicides such as L-phosphinothricin. The nucleotide sequence of aphosphinothricin-acetyl-transferase gene is provided in Europeanapplication No. 0 242 246 to Leemans et al., DeGreef et al.,Bio/Technology 7:61 (1989), describe the production of transgenic plantsthat express chimeric bar genes coding for phosphinothricin acetyltransferase activity. Exemplary of genes conferring resistance tophenoxy proprionic acids and cyclohexones, such as sethoxydim andhaloxyfop are the Acc1-S1, Acc1-S2 and Acc1-S3 genes described byMarshall et al., Theor. Appl. Genet. 83:435 (1992).

C. An herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) and a benzonitrile (nitrilase gene). Przibilla et al.,Plant Cell 3:169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441, and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J.285:173 (1992).

D. Acetohydroxy acid synthase, which has been found to make plants thatexpress this enzyme resistant to multiple types of herbicides, has beenintroduced into a variety of plants. See Hattori et al., Mol. Gen.Genet. 246:419, 1995. Other genes that confer tolerance to herbicidesinclude a gene encoding a chimeric protein of rat cytochrome P4507A1 andyeast NADPH-cytochrome P450 oxidoreductase (Shiota et al., PlantPhysiol., 106:17, 1994), genes for glutathione reductase and superoxidedismutase (Aono et al., Plant Cell Physiol. 36:1687, 1995), and genesfor various phosphotransferases (Datta et al., Plant Mol. Biol. 20:619,1992).

E. Protoporphyrinogen oxidase (protox) is necessary for the productionof chlorophyll, which is necessary for all plant survival. The protoxenzyme serves as the target for a variety of herbicidal compounds. Theseherbicides also inhibit growth of all the different species of plantspresent, causing their total destruction. The development of plantscontaining altered protox activity which are resistant to theseherbicides are described in U.S. Pat. Nos. 6,288,306; 6,282,837;5,767,373; and international publication WO 01/12825.

3. Genes That Confer or Contribute to a Value-Added Trait, Such as:

A. Increased iron content of the pepper, for example by transforming aplant with a soybean ferritin gene as described in Goto et al., ActaHorticulturae. 2000, 521, 101-109.

B. Increased sweetness of the pepper by transferring a gene coding formonellin that elicits a flavor 100,000 times sweeter than sugar on amolar basis. See Penarrubia et al., Biotechnology. 1992, 10: 561-564.

4. Genes that Control Male-Sterility

A. Introduction of a deacetylase gene under the control of atapetum-specific promoter and with the application of the chemicalN-Ac-PPT. See international publication WO 01/29237.

B. Introduction of various stamen-specific promoters. See internationalpublications WO 92/13956 and WO 92/13957.

C. Introduction of the barnase and the barstar genes. See Paul et al.,Plant Mol. Biol. 19:611-622, 1992).

Methods for Pepper Transformation

Numerous methods for plant transformation have been developed, includingbiological and physical, plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, GlickB. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88. In addition, expression vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993)pages 89-119.

A. Agrobacterium-mediated Transformation

One method for introducing an expression vector into plants is based onthe natural transformation system of Agrobacterium. See, for example,Horsch et al., Science 227:1229 (1985), Curtis et al., Journal ofExperimental Botany. 1994, 45: 279, 1441-1449, Torres et al., Plant cellTissue and Organic Culture. 1993, 34: 3, 279-285, Dinant et al.,Molecular Breeding. 1997, 3: 1, 75-86. A. tumefaciens and A. rhizogenesare plant pathogenic soil bacteria which genetically transform plantcells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes,respectively, carry genes responsible for genetic transformation of theplant. See, for example, Kado, C. I., Crit. Rev. Plant Sci. 10:1 (1991).Descriptions of Agrobacterium vector systems and methods forAgrobacterium-mediated gene transfer are provided by Gruber et al.,supra, Miki et al., supra, and Moloney et al., Plant Cell Reports 8:238(1989). See also, U.S. Pat. No. 5,591,616 issued Jan. 7, 1997.

B. Direct Gene Transfer

Several methods of plant transformation collectively referred to asdirect gene transfer have been developed as an alternative toAgrobacterium-mediated transformation. A generally applicable method ofplant transformation is microprojectile-mediated transformation whereinDNA is carried on the surface of microprojectiles measuring 1 to 4 μm.The expression vector is introduced into plant tissues with a biolisticdevice that accelerates the microprojectiles to speeds of 300 to 600 m/swhich is sufficient to penetrate plant cell walls and membranes.Russell, D. R., et al. PI. Cell. Rep. 12(3, January), 165-169 (1993),Aragao, F. J. L., et al. Plant Mol. Biol. 20 (2, October), 357-359(1992), Aragao, F. J. L., et al. PI. Cell. Rep. 12(9, July), 483-490(1993). Aragao, Theor. Appl. Genet. 93: 142-150 (1996), Kim, J.;Minamikawa, T. Plant Science 117: 131-138 (1996), Sanford et al., Part.Sci. Technol. 5:27 (1987), Sanford, J. C., Trends Biotech. 6:299 (1988),Klein et al., Bio/Technology 6:559-563 (1988), Sanford, J. C., PhysiolPlant 7:206 (1990), Klein et al., Biotechnology 10:268 (1992).

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., Bio/Technology 9:996 (1991). Alternatively,liposome and spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4:2731 (1985), Christouet al., Proc Natl. Acad. Sci. U.S.A. 84:3962 (1987). Direct uptake ofDNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol orpoly-L-ornithine has also been reported. Hain et al., Mol. Gen. Genet.199:161 (1985) and Draper et al., Plant Cell Physiol. 23:451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. Saker, M.; Kuhne, T. Biologia Plantarum 40(4): 507-514(1997/98), Donn et al., In Abstracts of VIIth International Congress onPlant Cell and Tissue Culture IAPTC, A2-38, p 53 (1990); D'Halluin etal., Plant Cell 4:1495-1505 (1992) and Spencer et al., Plant Mol. Biol.24:51-61 (1994). See also Chupean et al., Biotechnology. 1989, 7: 5,503-508.

Following transformation of pepper target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic line. The transgenic line could then be crossed,with another (non-transformed or transformed) line, in order to producea new transgenic pepper line. Alternatively, a genetic trait which hasbeen engineered into a particular pepper cultivar using the foregoingtransformation techniques could be moved into another line usingtraditional backcrossing techniques that are well known in the plantbreeding arts. For example, a backcrossing approach could be used tomove an engineered trait from a public, non-elite inbred line into anelite inbred line, or from an inbred line containing a foreign gene inits genome into an inbred line or lines which do not contain that gene.As used herein, “crossing” can refer to a simple X by Y cross, or theprocess of backcrossing, depending on the context.

Single-Gene Conversions

When the terms pepper plant, cultivar or pepper line are used in thecontext of the present invention, this also includes any single geneconversions of that line. The term “single gene converted plant” as usedherein refers to those pepper plants which are developed by a plantbreeding technique called backcrossing wherein essentially all of thedesired morphological and physiological characteristics of a cultivarare recovered in addition to the single gene transferred into the linevia the backcrossing technique. Backcrossing methods can be used withthe present invention to improve or introduce a characteristic into theline. The term “backcrossing” as used herein refers to the repeatedcrossing of a hybrid progeny back to one of the parental pepper plantsfor that line, backcrossing 1, 2, 3, 4, 5, 6, 7, 8 or more times to therecurrent parent. The parental pepper plant which contributes the genefor the desired characteristic is termed the nonrecurrent or donorparent. This terminology refers to the fact that the nonrecurrent parentis used one time in the backcross protocol and therefore does not recur.The parental pepper plant to which the gene or genes from thenonrecurrent parent are transferred is known as the recurrent parent asit is used for several rounds in the backcrossing protocol (Poehlman &Sleper, 1994; Fehr, 1987). In a typical backcross protocol, the originalcultivar of interest (recurrent parent) is crossed to a second line(nonrecurrent parent) that carries the single gene of interest to betransferred. The resulting progeny from this cross are then crossedagain to the recurrent parent and the process is repeated until a pepperplant is obtained wherein essentially all of the desired morphologicaland physiological characteristics of the recurrent parent are recoveredin the converted plant, in addition to the single transferred gene fromthe nonrecurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalline. To accomplish this, a single gene of the recurrent cultivar ismodified or substituted with the desired gene from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphological,constitution of the original line. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross, one ofthe major purposes is to add some commercially desirable, agronomicallyimportant trait to the plant. The exact backcrossing protocol willdepend on the characteristic or trait being altered to determine anappropriate testing protocol. Although backcrossing methods aresimplified when the characteristic being transferred is a dominantallele, a recessive allele may also be transferred. In this instance itmay be necessary to introduce a test of the progeny to determine if thedesired characteristic has been successfully transferred.

Many single gene traits have been identified that are not regularlyselected for in the development of a new line but that can be improvedby backcrossing techniques. Single gene traits may or may not betransgenic, examples of these traits include but are not limited to,male sterility, modified fatty acid metabolism, modified carbohydratemetabolism, herbicide resistance, nematode resistance, resistance forbacterial, fungal, or viral disease, insect resistance, enhancednutritional quality, industrial usage, yield stability and yieldenhancement. These genes are generally inherited through the nucleus.Several of these single gene traits are described in U.S. Pat. Nos.5,777,196, 5,948,957 and 5,969,212, the disclosures of which arespecifically hereby incorporated by reference.

Tissue Culture

Further reproduction of the variety can occur by tissue culture andregeneration. Tissue culture of various tissues of pepper andregeneration of plants therefrom is well known and widely published. Forexample, reference may be had to Teng et al., HortScience. 1992, 27: 9,1030-1032 Teng et al., HortScience. 1993, 28: 6, 669-1671, Zhang et al.,Journal of Genetics and Breeding. 1992, 46: 3, 287-290, Webb et al.,Plant Cell Tissue and Organ Culture. 1994, 38: 1, 77-79, Curtis et al.,Journal of Experimental Botany. 1994, 45: 279, 1441-1449, Nagata et al.,Journal for the American Society for Horticultural Science. 2000, 125:6, 669-672, and Ibrahim et al., Plant Cell, Tissue and Organ Culture.(1992), 28 (2): 139-145. It is clear from the literature that the stateof the art is such that these methods of obtaining plants are routinelyused and have a very high rate of success. Thus, another aspect of thisinvention is to provide cells which upon growth and differentiationproduce pepper plants having the physiological and morphologicalcharacteristics of the hybrid ‘E 499526’.

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, meristematic cells, andplant cells that can generate tissue culture that are intact in plantsor parts of plants, such as leaves, pollen, embryos, roots, root tips,anthers, pistils, flowers, seeds, petioles, and the like. Means forpreparing and maintaining plant tissue culture are well known in theart. By way of example, a tissue culture comprising organs has been usedto produce regenerated plants. U.S. Pat. Nos. 5,959,185; 5,973,234 and5,977,445 describe certain techniques, the disclosures of which areincorporated herein by reference.

Additional Breeding Methods

This invention also is directed to methods for producing a pepper plantby crossing a first parent pepper plant with a second parent pepperplant wherein the first or second parent pepper plant is a pepper plantof cultivar ‘E 499526’. Further, both first and second parent pepperplants can come from pepper cultivar ‘E 499526’. Thus, any such methodsusing pepper cultivar ‘E 499526’ are part of this invention: selfing,backcrosses, hybrid production, crosses to populations, and the like.All plants produced using pepper cultivar ‘E 499526’ as at least oneparent are within the scope of this invention, including those developedfrom cultivars derived from pepper cultivar ‘E 499526’. Advantageously,this pepper cultivar could be used in crosses with other, different,pepper plants to produce the first generation (F₁) pepper hybrid seedsand plants with superior characteristics. The cultivar of the inventioncan also be used for transformation where exogenous genes are introducedand expressed by the cultivar of the invention. Genetic variants createdeither through traditional breeding methods using pepper cultivar ‘E499526’ or through transformation of cultivar ‘E 499526’ by any of anumber of protocols known to those of skill in the art are intended tobe within the scope of this invention.

The following describes breeding methods that may be used with pepperhybrid ‘E 499526’ in the development of further pepper plants. One suchembodiment is a method for developing progeny pepper plants in a pepperplant breeding program comprising: obtaining the pepper plant, or a partthereof, of cultivar ‘E 499526’, utilizing said plant or plant part as asource of breeding material, and selecting a pepper cultivar ‘E 499526’progeny plant with molecular markers in common with cultivar ‘E 499526’and/or with morphological and/or physiological characteristics selectedfrom the characteristics listed in Table 1. Breeding steps that may beused in the pepper plant breeding program include pedigree breeding,backcrossing, mutation breeding, and recurrent selection. In conjunctionwith these steps, techniques such as RFLP-enhanced selection, geneticmarker enhanced selection (for example SSR markers) and the making ofdouble haploids may be utilized.

Another method involves producing a population of pepper cultivar ‘E499526’ progeny pepper plants, comprising crossing cultivar ‘E 499526’with another pepper plant, thereby producing a population of pepperplants, which, on average, derive 50% of their alleles from peppercultivar ‘E 499526’. A plant of this population may be selected andrepeatedly selfed or sibbed with a pepper cultivar resulting from thesesuccessive filial generations. One embodiment of this invention is thepepper cultivar produced by this method and that has obtained at least50% of its alleles from pepper cultivar‘E 499526’.

One of ordinary skill in the art of plant breeding would know how toevaluate the traits of two plant varieties to determine if there is nosignificant difference between the two traits expressed by thosevarieties. For example, see Fehr and Walt, Principles of CultivarDevelopment, p 261-286 (1987). Thus the invention includes peppercultivar ‘E 499526’ progeny pepper plants comprising a combination of atleast two cultivar ‘E 499526’ traits selected from the group consistingof those listed in Table 1 or the cultivar ‘E 499526’ combination oftraits listed in the Summary of the Invention, so that said progenypepper plant is not significantly different for said traits than peppercultivar ‘E 499526’ as determined at the 5% significance level whengrown in the same environmental conditions. Using techniques describedherein, molecular markers may be used to identify said progeny plant asa pepper cultivar ‘E 499526’ progeny plant. Mean trait values may beused to determine whether trait differences are significant, andpreferably the traits are measured on plants grown under the sameenvironmental conditions. Once such a variety is developed its value issubstantial since it is important to advance the germplasm base as awhole in order to maintain or improve traits such as yield, diseaseresistance, pest resistance, and plant performance in extremeenvironmental conditions.

Progeny of pepper cultivar ‘E 499526’ may also be characterized throughtheir filial relationship with pepper cultivar ‘E 499526’, as forexample, being within a certain number of breeding crosses of peppercultivar ‘E 499526’. A breeding cross is a cross made to introduce newgenetics into the progeny, and is distinguished from a cross, such as aself or a sib cross, made to select among existing genetic alleles. Thelower the number of breeding crosses in the pedigree, the closer therelationship between pepper cultivar ‘E 499526’ and its progeny. Forexample, progeny produced by the methods described herein may be within1, 2, 3, 4 or 5 breeding crosses of pepper cultivar ‘E 499526’.

As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cell tissue cultures from which pepper plants can beregenerated, plant calli, plant clumps and plant cells that are intactin plants or parts of plants, such as fruit, leaves, pollen, embryos,cotyledons, hypocotyl, roots, root tips, anthers, pistils, flowers,seeds, stems and the like.

TABLES

In Table 2 that follows the morphologic and other characteristics ofanother hybrid pepper cultivar is shown. ‘E 499524’ is a hybrid peppercultivar with high yield potential. It is a medium vigorous plant thatproduces medium green immature fruit and light red mature fruit of avery small size (jalapeno pepper size) but mild flavor (not hot). Inaddition, the plants are suitable for covered cultivation and open fieldgrowing. The hybrid cultivar has shown uniformity and stability for thetraits, within the limits of environmental influence for the traits. Thecultivar has been increased with continued observation for uniformity.No variant traits have been observed or are expected in ‘E 499524’.

TABLE 2 VARIETY DESCRIPTION INFORMATION FOR E 499524 GENERAL: Type:Sweetbite Usage: Fresh market Type of culture: Covered cultivation andopen field PLANT: Seedling, anthocyanin coloration of hypocotyl: PresentShortened internode: Absent Vigor: Medium Flower, attitude of peduncle:Non-erect FRUIT: Color before maturity: Green Intensity of color beforematurity: Medium Length: 5 cm Diameter: 2 cm Predominant shape oflongitundinal section: Trapezoid Color at maturity: Red Intensity ofcolor at maturity: Light Predominant number of locules: Two and threeCapsaicin in placenta: Absent Time of ripening (color change of fruitson 50% of plants): Medium DISEASE Tobamovirus pathotype P₀: ResistantRESISTANCE: Tobamovirus pathotype P₁: Susceptible Tobamovirus pathotypeP₁₋₁: Susceptible Tobamovirus pathotype P₁₋₂₋₃: Susceptible Potato VirusY pathotype P₀: Susceptible Potato Virus Y pathotype P₁: SusceptiblePotato Virus Y pathotype P₁₋₂: Susceptible Phytophthora capsici:Susceptible Tomato Spotted Wilt Virus: Susceptible Cucumber MosaicVirus: Susceptible

In Table 3 that follows the morphologic and other characteristics ofanother hybrid pepper cultivar is shown. ‘E 499531’ is a hybrid peppercultivar with high yield potential. It is a medium vigorous plant thatproduces medium green immature fruit and medium orange mature fruit of avery small size (jalapeno pepper size) but mild flavor (not hot). Inaddition, the plants are suitable for covered cultivation and open fieldgrowing. The hybrid cultivar has shown uniformity and stability for thetraits, within the limits of environmental influence for the traits. Thecultivar has been increased with continued observation for uniformity.No variant traits have been observed or are expected in ‘E 499531’.

TABLE 3 VARIETY DESCRIPTION INFORMATION FOR E 499531 GENERAL: Type:Sweetbite Usage: Fresh market Type of culture: Covered cultivation andopen field PLANT: Seedling, anthocyanin coloration of hypocotyl: PresentShortened internode: Absent Vigor: Medium Flower, attitude of peduncle:Non-erect FRUIT: Color before maturity: Green Intensity of color beforematurity: Medium Length: 4 cm Diameter: 2 cm Predominant shape oflongitundinal section: Trapezoid Color at maturity: Orange Intensity ofcolor at maturity: Medium Predominant number of locules: Two and threeCapsaicin in placenta: Absent Time of ripening (color change of fruitson 50% of plants): Medium DISEASE Tobamovirus pathotype P₀: ResistantRESISTANCE: Tobamovirus pathotype P₁: Susceptible Tobamovirus pathotypeP₁₋₁: Susceptible Tobamovirus pathotype P₁₋₂₋₃: Susceptible Potato VirusY pathotype P₀: Susceptible Potato Virus Y pathotype P₁: SusceptiblePotato Virus Y pathotype P₁₋₂: Susceptible Phytophthora capsici:Susceptible Tomato Spotted Wilt Virus: Susceptible Cucumber MosaicVirus: Susceptible

Deposit Information

A deposit of the hybrid pepper of this invention is maintained by EnzaZaden, Salinas, California. Access to this deposit will be availableduring the pendency of this application to persons determined by theCommissioner of Patent and Trademarks to be entitled thereto under 37CRF 1.14 and 35 USC 122. Upon allowance of any claims in thisapplication, all restrictions on the availability to the public of thevariety will be irrevocably removed by affording access to a deposit ofat least 2,500 seeds of the same variety with the American Type CultureCollection (ATCC), 10801 University Boulevard, Manassas, Va. 20110 orNational Collections of Industrial, Food and Marine Bacteria (NCIMB), 23St Machar Drive, Aberdeen, Scotland, AB24 3RY, United Kingdom.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions, and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions, and sub-combinations as are within their truespirit and scope.

1. A hybrid pepper seed designated ‘E 499526’ wherein a representativesample of seed has been deposited under ATCC Accession No. ______.
 2. Apepper plant produced by growing the seed of claim
 1. 3. Pollen, ovule,seed, or fruit, of the plant, or a part of the plant, of claim
 2. 4. Apepper plant propagated from one or more of the plant parts of claim 3.5. A pepper plant having all of the physiological and morphologicalcharacteristics of the pepper plant of claim
 2. 6. Pollen, ovule, seed,or fruit, of the plant, or a part of the plant, of claim
 5. 7. A pepperplant propagated from one or more of the plant parts of claim
 6. 8. ASweetbite pepper with a diameter between 1.5 centimeters and 3centimeters and a length between 3 centimeters and 7 centimeters. 9.Pollen, ovule, seed, or fruit, of the plant, or a part of the plant, ofclaim
 8. 10. A pepper plant propagated from one or more of the plantparts of claim
 9. 11. A tissue culture of cells produced from the pepperplant of claims 2, 5 and 8, wherein said cells of the tissue culture areproduced from a plant part selected from the group consisting of leaf,anther, pistil, stem, petiole, root, root tip, fruit, seed, flower,cotyledon, hypocotyl, embryo and meristematic cell.
 12. A pepper plantregenerated from tissue culture of claim
 11. 13. Pollen, ovule, seed, orfruit, of the plant, or a part of the plant, of claim
 12. 14. Aprotoplast produced from the plant of claims 2, 5 and
 8. 15. A pepperregenerated from the protoplast of claim
 14. 16. Pollen, ovule, seed, orfruit, of the plant, or a part of the plant, of claim
 15. 17. A methodfor producing a pepper plant comprising crossing the pepper plant ofclaims 2, 5 and 8 with a different pepper plant or with themselves andharvesting the resultant pepper seed.
 18. A pepper plant produced by themethod of claim
 17. 19. Pollen, ovule, seed, or fruit, of the plant, ora part of the plant, of claim
 18. 20. A method of producing a pepperplant wherein the method comprises transforming the pepper plant ofclaims 2, 5 and 8 with a transgene.
 21. A pepper plant produced by themethod of claim
 20. 22. Pollen, ovule, seed, or fruit, of the plant, ora part of the plant, of claim 21.